Elevated AST/ALT ratio is associated with all-cause mortality in patients with stable coronary artery disease: a secondary analysis based on a retrospective cohort study

The aim of this study is to explore the association between the aspartate amino transferase (AST)/alanine aminotransferase (ALT) ratio and all-cause mortality (ACM) in stable coronary artery disease (CAD) patients treated by percutaneous coronary intervention (PCI). The study is a secondary analysis of a retrospective cohort study involving 203 stable CAD patients. Patients were divided into two groups, based on the optimal AST/ALT ratio threshold calculated by the ROC curve (low group: AST/ALT ratio < 1.40; high group: AST/ALT ratio ≥ 1.40). Results were compared using hazard ratio (HR) and a 95% confidence interval (CI). ACM occurred in 18 patients after an average follow-up time of 749 (435–1122) days. Among them, ACM occurred in 6 patients in the low group and 12 patients in the high group, with significant differences between the groups (4.65% versus 16.22%, P value = 0.005). In the Kaplan–Meier analysis, an elevated AST/ALT ratio was associated with increased ACM in stable ACD patients (HR 3.78, 95% CI 1.44–9.93, P value < 0.001). An elevated AST/ALT ratio was still found to be an independent prognostic factor for ACM (HR 2.93, 95% CI 1.08–7.91, P value = 0.034) after adjusting for potential confounders. Therefore, an elevated AST/ALT ratio is an independent prognostic factor for ACM in stable ACD patients.

Definitions and endpoints. As described in Suzuki's study 18 , therapeutic clinicians use all available information, including symptoms, laboratory results, electrocardiograms, echocardiography, and coronary angiography, to accurately diagnose old myocardial infarction. Stable CAD was defined as having ≥ 90% angiographic evidence of epicardial coronary artery stenosis, or ≥ 75% angiographic evidence of epicardial coronary artery stenosis. Occurrence of chest pain symptoms or stress ischemia induced by exercise, or any clinical stress test pattern is also included. Patients were given aspirin and thienopyridines pre-operation. Cardiologists performed coronary angiography and PCI according to standard protocols and guidelines. All-cause mortality (ACM) is the primary endpoint in this study.
Statistical analysis. Firstly, since the goal of this study is to explore the association between the AST/ALT ratio and ACM in stable CAD patients, the normality of the AST/ALT ratio is initially assessed via a histogram. Secondly, the receiver operating characteristic (ROC) curve was used to explore the diagnostic value and optimal threshold of the AST/ALT ratio for ACM. The patients were subsequently divided into two groups according to the optimal threshold. Meanwhile, the sensitivity, specificity, and Youden index of the AST/ALT ratio in diagnosing ACM were recorded.
Continuous variables were represented by the mean and standard deviation (SD), and differences between groups were compared by an independent sample t-test. Categorical variables were represented by cases and percentages, and differences between groups were compared via chi-squared test. Kaplan-Meier analysis, univariate, and multivariate Cox proportional hazard regression analysis were also used to estimate the influence of the AST/ALT ratio on ACM. Results were compared using hazard ratio (HR) and a 95% confidence interval (CI).
In this study, the AST/ALT ratio was considered as the independent variable, ACM was considered as the dependent variable, and other variables were considered as covariates. The following approaches were used to screen covariates. Firstly, univariate Cox proportional hazard regression analysis was used. Factors with a P-value of < 0.05 indicate that this covariate may influence the results. Secondly, if the effect of covariates on the AST/ALT ratio regression coefficient is less than 10%, those covariates are eliminated from multivariate Cox proportional hazard regression analysis 21 . Otherwise, the covariates are included in the subsequent analysis. Thirdly, considering the available case of events, the covariates to be included are carefully selected to ensure the parsimony of the ultimate model 22,23 . Furthermore, considering the possibility of multicollinearity among variables 24 , Pearson correlation analysis was performed to identify the correlation between variables. If a variable is highly correlated with the AST/ALT ratio, those covariates are excluded from the Cox proportional hazard regression analysis 25 .

Results
AST/ALT ratio distribution. AST/ALT was missed in one patient, and the corresponding AST/ALT ratio could not be calculated therefore the patient was excluded from this study. 203 patients were actually included. The histogram in Fig. 1 indicates that AST/ALT ratio conforms to a skewness distribution. The AST/ALT ratio median, minimum, and maximum are 1.23, 0.52, and 4.80, respectively. Study population. Patients were divided into two groups, based on the optimal threshold (1.40) of the AST/ALT ratio: low group (AST/ALT ratio < 1.40, N = 129) and high group (AST/ALT ratio ≥ 1.40, N = 74). The baseline characteristics of both groups are shown in Table 1. As also shown in Table 1, patients in the high group were older and had lower hemoglobin, albumin, estimated glomerular filtration rate (eGFR), triglycerides, and statin use, compared with the low group. On the other hand, the proportion of patients with a normal body mass index (BMI) was higher in the high group than in the low group. No significant differences in other variables were observed between the two groups. ACM occurred in 18 patients after an average follow-up time of 749 (435-1122) days. Among them, ACM occurred in 6 patients in the low group and 12 patients in the high group, with significant differences between groups (4.65% versus 16.22%, P value = 0.005, Fig. 3).
Univariate Cox proportional hazard regression analysis associated with ACM. In Table 2, univariate Cox proportional hazard regression analysis indicated that advanced age (HR 5.04, 95% CI 1.16-21.96) and AST/ALT ratio (HR 3.56, 95% CI 1.33-9.51) were associated with a higher risk of ACM, while past smoking  www.nature.com/scientificreports/ Lasso regression analysis associated with ACM. Variables, including age, AST/ALT ratio, past smoking, hemoglobin, albumin, eGFR, total cholesterol, LDL, aspirin, and statins, selected from univariate Cox analysis, were incorporated into Lasso regression analysis to further screen out variables associated with ACM. The Lasso regression analysis revealed that when the minimum mean square error λ is 0.017, the model is optimal. At this time, the identified variables are age, AST/ALT ratio, hemoglobin, albumin, total cholesterol, and LDL (Fig. 4). Fig. 5. In the Kaplan-Meier analysis, an elevated AST/ALT ratio is associated with increased ACM in stable ACD patients (HR 3.78, 95% CI 1.44-9.93, P < 0.001) after an average follow-up time of 749 (435-1122) days.

Multivariate Cox proportional hazard regression analysis.
Based on the above covariate screening criteria, the influence of age, total cholesterol, and LDL, on the AST/ALT ratio regression coefficient is less than 10%, therefore these covariables can be excluded. Furthermore, Suzuki's 18 study proposes that albumin is associated with the prognosis of stable CAD patients, considering the high correlation between hemoglobin and the AST/ALT ratio. Consequently, to ensure the simplicity of the model, albumin was adjusted as a confounding factor in the subsequent analysis. After adjusting for potential confounders using multivariate Cox regression analysis, an elevated AST/ALT ratio was still found to be an independent prognostic factor for ACM in stable ACD patients (HR 2.93, 95% CI 1.08-7.91, P value = 0.034, Fig. 6). This indicates that a higher AST/ALT ratio is associated with a higher risk of ACM.

Discussion
The main findings of this retrospective cohort study, based on long-term follow-up, are as follows: firstly, to the best of our knowledge, this study is the first to use AST/ALT ratio to assess outcomes in stable CAD patients. It is found that the AST/ALT ratio is an important serological marker that can be used to predict ACM in stable CAD patients. The optimal prediction threshold is 1.4, and the sensitivity and specificity are 66.67% and 66.49%, respectively. Secondly, after adjusting for potential confounding factors, the AST/ALT ratio is a prognostic risk factor for ACM in stable CAD patients. This suggests that the AST/ALT ratio could be used in clinical practice to further refine risk stratification for stable CAD patients. Furthermore, the results of this study suggest that if the AST/ALT ratio is measured at admission, patients at high risk of ACM can be identified early. Therefore, clinicians should focus on such high-risk populations during follow-up. It is acknowledged that the AST/ALT ratio is an important blood circulation biomarker. Most previous studies on the AST/ALT ratio propose that the AST/ALT ratio level is related to the prognosis of patients suffering from different diseases [15][16][17] . However, few studies have stated that the AST/ALT ratio is not related to disease 26 . A retrospective cohort study involving 380 patients with toxic hepatitis revealed that AST/ALT ratios could not predict the severity of liver fibrosis 26 . AST is found to be highly expressed in the brain, muscle, and kidney tissues, while ALT is considered to have higher liver specificity or abundant expression in liver tissues 27 . Pathological states can lead to tissue damage and higher abnormal status, resulting in a significant increase in AST level rather than ALT, making the AST/ALT ratio an attractive potential marker 28,29 . This study also found that the AST/ALT ratio was related to stable CAD patient prognosis, that is, an elevated AST/ALT ratio is associated with ACM in stable CAD patients. After adjusting for potential confounding factors using multivariate Cox regression analysis, the relationship between the AST/ALT ratio and ACM still exists (HR 2.93, 95% CI 1.08-7.91). This indicates that the AST/ALT ratio can serve as a serological marker to quantify risk stratification in stable CAD patients. www.nature.com/scientificreports/ Our results are consistent with previous studies regarding the AST/ALT ratio and cardiovascular disease. A large sample cohort study with 14,220 participants examined the association between the AST/ALT ratio with ACM and cardiovascular mortality in hypertensive patients. This study found that an increased AST/ALT ratio was associated with increased risk of ACM (P value < 0.05) and cardiovascular mortality (P value < 0.05) in hypertensive patients 30 , after an average follow-up time of 1.7 years. Another study, based on the general population, followed 3494 participants for up to 10 years and found that the AST/ALT ratio increased with brain natriuretic www.nature.com/scientificreports/ peptide (BNP). Furthermore, multivariate regression analysis suggested that a higher AST/ALT ratio was an independent predictor for ACM and cardiovascular mortality, and the authors further proposed that the AST/ ALT ratio is a simple and low-cost mortality predictor 15,31 .
Possible explanations for an elevated AST/ALT ratio leading to a higher risk of ACM in stable CAD patients are as follows: previous studies have shown that high AST activity is associated with higher mortality 32,33 , and low ALT levels are associated with aging, frailty, and higher mortality, in elderly populations 34 . Furthermore, previous studies have demonstrated that the main reason for the reduction in ALT is the significant reduction in liver size and hepatic blood flow 31,35 , and patients with a history of cardiovascular disease tend to have lower ALT levels. It is speculated that low hepatic blood flow may be related to underlying myocardial injury. This phenomenon, known as cardiohepatic syndrome 36,37 , proposes that the heart and liver interact to exacerbate or slow disease progress. Considering these cardio-hepatic interactions, clinical practice should not only focus on the improvement of one particular organ but should focus on the functional improvement of all organs. Moreover, elevated transaminases in clinical practice may lead to a reduction in statin use, which may partially explain the increased risk of developing ACM.  www.nature.com/scientificreports/ This study has some limitations. Firstly, there may be potential bias inherent in retrospective studies. Secondly, this study is a secondary analysis based on a retrospective study, and its participants may have undetected liver, or other, disease that affects the serum AST or ALT levels, leading to possible confounding bias in the AST/ ALT ratio. Thirdly, the subjects of this study were patients from a single center, and caution should be exercised when applying this result to other ethnic groups. Fourthly, the patients included in this study were Japanese, and further evidence is needed to verify whether it is applicable to other populations. Further prospective, large sample, multi-center studies are needed to validate the results of this research.

Conclusion
An elevated AST/ALT ratio is an independent prognostic factor for ACM in stable ACD patients. In clinical practice, the risk stratification of stable CAD patients could be refined by determining the AST/ALT ratio concentration, thereby improving their short-and long-term prognoses. In addition, considering that the AST/ALT ratio is an inexpensive routine serological marker in clinical practice, it has the potential for further promotion and application.